The analysis of nanoparticles is a ubiquitous requirement in a broad range of industry sectors. Product performance and stability frequently depends on the ability to manufacture particle suspensions to fine tolerances without the presence of contaminants or aggregates. Foremost in such analyses is particle size and size distribution measurement for which a number of techniques are well established and commonly employed in routine quality control as well as in a research and development environment. Depending on the nature of the product and the particle characteristics sought, one or more of a range of analytical methodologies are employed which include electron microscopy, dynamic light scattering (DLS), Fraunhofer scattering, single particle detection techniques, optical microscopy, etc. For particles in the nanoscale, however, only the first two of these examples are used frequently. While widespread, both have drawbacks including capital and running costs, analysis turnaround time, and, in the case of dynamic light scattering, a limited ability to resolve particle size distribution profiles.
Dynamic Light Scattering (DLS)
Dynamic light scattering techniques such as photon correlation spectroscopy (PCS) analyse a large ensemble of many thousands of particles from which only a z-average particle diameter, i.e. intensity weighted particle mean diameter, is obtained as well as a polydispersity quotient indicating the width of the particle size distribution (Pecora, R., (Ed)(1985) Dynamic Light Scattering, Applications of Photon Correlation Spectroscopy, Plenum Press, New York). The technique is typically practiced on a sample comprising a suspension of nanoparticles in a liquid, illuminated by a suitably focussed coherent, monochromatic laser beam of approximately 100 microns diameter, the light scattered from which is detected by a photon counting photomultiplier. The detector is configured, by means of a pinhole and slit combination or single mode fibre optic, to observe only a single coherence area or speckle from the light scattered by the sample into the far field. The intensity of light within the coherence area fluctuates through interference effects as a consequence of random Brownian motion of the nanoparticles and the characteristic timescale of the intensity fluctuations are analysed by a digital correlator. The average rate of change of the intensity fluctuations can be expressed in terms of particle diffusion coefficient (Dt) from which a sphere equivalent hydrodynamic diameter of particles in the path of the laser beam can be estimated. The maximum dimension, e.g. diameter, of particles as small as 2-3 nm can be determined in this way. However, as all particles are measured simultaneously in DLS, it is frequently the case that a relatively small number of highly scattering larger particles (e.g. contaminants or aggregates) can dominate the signal and effectively obscure the presence of the bulk of the smaller particles that may be present. In some limited circumstances it is possible, through the application of various de-convolution algorithms, to extract particle size distribution structure (e.g. a bimodal distribution) from the results obtained but this approach is reliable only if the two populations are not too polydisperse themselves or too close together in size. In practice, particles which differ in diameter by less than a ratio of 3 cannot normally be resolved. This represents a severe limitation in applications in which accurate information about particle size distribution is required, but the samples contain larger particles (e.g. contaminants or aggregates) which significantly bias the results and can partially or totally mask the presence of smaller particles. Equally, inherently complex polydisperse and heterogeneous samples containing a broad range of particle sizes generate distributions which are frequently badly skewed to the larger particle sizes present. Finally, being an ensemble technique, no direct information concerning the numbers of any particular particle size or size class can be recovered from DLS.
Nanoparticle Tracking Analysis (NTA)
Nanoparticle tracking analysis is a recently developed method for the direct and realtime visualisation and analysis of nanoparticles in liquids. See e.g. WO 03/093801. Based on a laser illuminated microscopical technique, Brownian motion of nanoparticles is analysed in real-time by a charge-couple device (CCD) camera, each particle being simultaneously but separately visualised and tracked by a dedicated particle tracking image-analysis programme. Because each particle is visualised and analysed separately, the resulting estimate of particle size and particle size distribution does not suffer from the limitation of being an intensity weighted, z-average distribution, which is the norm in conventional ensemble methods of particle sizing in this size regime, e.g. dynamic light scattering (DLS) as described above. The ability of NTA to measure simultaneously particle size and particle scattering intensity allows heterogeneous particle mixtures to be resolved and, importantly, particle concentration to be estimated directly, the particle size distribution profile obtained by NTA being a direct number/frequency distribution.
NTA has become a term of art, recognised by those skilled in the relevant field. There are over 250 scientific papers and presentations referring to data collected using NTA. Further the term is used by, for example, ASTM International (formerly the American Society for Testing and Materials), the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) and the NIH.
The range of particle sizes that can be analysed by NTA depends on the particle type. The lower size limit is defined by the particle size and particle refractive index given that sufficient light must be scattered by each particle for it to be detected and tracked as described above. For particles with very high refractive indices, such as colloidal gold, accurate determination of size can be achieved down to particles with a maximum dimension of about 10 nm. For lower refractive index particles, such as those of biological origin, the smallest detectable size might be in the range 25-50 nm Accordingly, NTA is limited by its ability to detect particles below a certain size.
With NTA, the presence and analysis of particles, each of which scatters sufficient light to be detected individually, can still be carried out even in the presence of ‘background’ material comprising, for instance, a population of very small particles (such as protein molecules, sub-10 nm inorganic material, polymer solutions, nano-emulsions, etc.) each of which is too small to detect individually but which is present in sufficiently high concentration to collectively form a background haze of scattered light. This background cannot be analysed by NTA but particles visible as discrete light scattering entities embedded within in this background may be analysed by NTA. Of course, the intensity of this background will determine the limit of sensitivity of NTA in terms of minimum detectable size. Further, NTA is able to identify, track and analyse suitably sized particles even when they are present in heterogeneous samples containing high numbers of larger particles.
NTA is further capable of detecting and analysing fluorescent or fluorescently labelled nanoparticles in the presence of a non-fluorescent background though use of appropriate fluorescence exciting optical sources and suitable fluorescence filters. NTA is further capable of measuring more than one fluorescence wavelength within a sample using multiple filters or a colour camera.
Thus, DLS can serve the requirements of analysis of nanoparticle size down to sizes of 2-3 nm but suffers badly from an intensity weighting to larger particles (such as contaminants or aggregates) in the sample and cannot furnish information about particle number, whereas NTA can detect, analyse and count individual particles down to sizes of, say, 10-50 nm but cannot detect and analyse particles below this size limit which, if present, appear as a background haze. NTA-detectable particles are referred to herein as relatively larger particles or contaminant particles, and smaller particles, not detectable by NTA, are referred to herein as relatively smaller particles, haze or background.